Project Summary CRISPR-Cas systems are recently discovered RNA-based adaptive immune systems that control invasions of viruses and other mobile genetic elements in prokaryotes. CRISPR-Cas systems function by capturing short invader sequences within the CRISPR locus of the host genome, producing short crRNAs from the CRISPR locus transcripts, and using the crRNAs to guide Cas protein-containing immune effector complexes to recognize and destroy the invading nucleic acids. CRISPR-Cas based immunity is mediated by numerous and diverse Cas proteins and a given organism may possess one or more of the twelve distinct sets of known CRISPR-Cas immune modules. We currently know very little about how the key steps in the fascinating CRISPR-Cas immune response pathways occur for most of the systems. Using a powerful combination of molecular, genetic, structural, and biochemical approaches, we will determine the molecular basis for how various CRISPR-Cas systems acquire foreign DNA sequence in their CRISPR locus memory banks to provide heritable immunity against specific invaders. We will also delineate the mechanisms by which diverse crRNA- Cas protein immune effector complexes selectively recognize and destroy foreign nucleic acids as a means to combat the viruses and other transgressors. A comprehensive understanding of how the structurally and functional diverse CRISPR-Cas immune systems each function is essential toward understanding the range of mechanisms that have evolved to protect multitudes of prokaryotes from potentially lethal viral attack. The knowledge gained by our research program will contribute directly to ongoing efforts aimed at exploiting CRISPR-Cas systems as powerful research tools for genome editing and controlled gene expression as well as novel CRISPR-based, sequence-specific antibiotics to selectively target human pathogens (bacteria and viruses) and limit the spread of antibiotic resistance.